小鼠巨噬细胞系体外感染卡介苗的应答

【目的】探讨小鼠巨噬细胞系RAW264.7体外感染卡介苗的应答。【方法】体外感染RAW264.7细胞23h后,分析细胞形态和细胞表面共刺激分子的表达。然后去除培养上清中的卡介苗,继续培养不同时间,通过CFSE、annexin V/PI和Rh123标记,分析宿主细胞的应答。【结果】卡介苗感染23h后,细胞生长状态良好,细胞内能明显观察到吞噬泡中的BCG。细胞表面共刺激分子CD40、CD54、CD80、CD86、CD11b的表达明显升高,CD11c、I-Ad以及H-2Kd的表达变化不明显。CFSE标记卡介苗后,随着培养时间的延长,荧光强度逐渐减弱,但是4天后仍然明显地高于对照组。除去培养上清中的卡介苗后继续培养,含有卡介苗的细胞逐渐减少,继续培养60h后基本检测不到。另外,卡介苗感染不能诱导细胞凋亡,线粒体膜电位先升高后降低,5d后,基本上与对照组一致。【结论】通过以上分析,为卡介苗免疫机理的研究提供了重要数据。     

LncRNA-GAS5对BCG感染小鼠巨噬细胞后细胞坏死的调控作用

【背景】LncRNA-GAS5是由Gas5基因编码的功能性LncRNA分子,对细胞极化、细胞凋亡、坏死和自噬等多种生物学过程具有重要的调控作用。【目的】探讨LncRNA-GAS5对卡介苗(Bacillus Calmette-Guérin,BCG)诱导的小鼠巨噬细胞RAW264.7坏死的调控作用。【方法】构建LncRNA-GAS5的过表达及干扰载体,分别转染巨噬细胞RAW264.7后使用BCG感染,采用噻唑蓝比色法(MTT)检测细胞存活率,通过透射电镜观察细胞坏死形态,利用碘化丙啶(Propidine iodide,PI)单染法流式细胞仪检测细胞坏死率,通过实时荧光定量PCR和Western blot法研究坏死相关调控因子RIP1、RIP3和MLKL的表达水平。【结果】BCG感染巨噬细胞后,LncRNA-GAS5表达水平显著上调;同时,采用LncRNA-GAS5过表达载体单独转染或结合BCG感染后,巨噬细胞存活率均下降且细胞坏死率显著增加。同时坏死关键调控因子RIP1、RIP3和MLKL的mRNA及蛋白表达水平均显著上调(P0.001),而GAS5干扰载体可有效抑制这一效果。【结论】LncRNA-GAS5通过上调坏死相关因子RIP1、RIP3及MLKL的表达促进BCG感染巨噬细胞后诱导的细胞坏死,研究结果为进一步探讨LncRNA-GAS5对BCG感染巨噬细胞后细胞坏死调控的分子机制奠定了基础。     

小鼠巨噬细胞RAW264.7 的培养技巧及经验总结

RAW264.7细胞具有很强的黏附和吞噬抗原的能力,是研究微生物学、免疫学的常用细胞株.很多研究者发现这种细胞形态极不稳定,细胞状态的评价也很困难.本文作者结合RAW264.7培养经历及文献资料探讨RAW264.7细胞培养的经验教训和评价细胞状态的方法,旨在为培养该细胞的科研工作者提供一定的借鉴.     

迷迭香酸抗穿透支原体脂蛋白诱导巨噬细胞凋亡的作用研究

探讨迷迭香酸对穿透支原体脂蛋白诱导巨噬细胞凋亡的保护作用。方法:以穿透支原体脂蛋白损伤RAW264．7细胞为模型,用MTT法检测细胞存活状况,DNA片断化分析观察穿透支原体脂蛋白对RAW264．7细胞DNA降解的影响,碘化丙啶染色流式细胞术检测细胞凋亡率。结果:7．5mg/L穿透支原体脂蛋白可引起RAW264．7细胞存活率降低,出现细胞凋亡特征性“DNA梯带”,流式细胞术检测细胞出现凋亡亚G1期峰,凋亡率为31．9%;100μmol/L迷迭香酸预处理1小时后可以升高RAW264．7细胞的存活率,凋亡细胞特征性的梯状梯带消失,细胞凋亡亚G1期峰消失,并使RAW264．7细胞的凋亡率下降为7．9%。结论:迷迭香酸有抗穿透支原体脂蛋白诱导RAW264．7细胞凋亡的作用。     

草木犀石油醚提取物对小鼠巨噬细胞促炎介质的影响

目的研究草木犀石油醚提取物在体外的抗炎作用。方法采用小鼠巨噬细胞系RAW264．7建立炎症细胞模型,加入10μg/L的LPS培养液和不同浓度的草木犀石油醚提取物进行干预。ELISA法检测上清液中TNF-α,IL-1β,IL-6和NO的分泌量;实时荧光定量RT-PCR检测TNF-α,iNOS和COX-2的mRNA表达;Western印迹法检测COX-2蛋白的表达。结果草木犀提取物干预后细胞所分泌的炎性介质（TNF—α,IL-1β,IL-6和NO）与模型组相比均显著降低（P〈0．01）,并存在剂量依赖关系;RT-PCR结果显示干预后细胞TNF-α,iNOS和COX-2的mRNA表达水平显著降低（P〈0．01）,也存在剂量依赖关系;Western印迹结果显示草木犀石油醚提取物及地塞米松干预后COX-2蛋白水平明显降低（P〈0．01）。结论草木犀的石油醚提取物通过下调LPS诱导的巨噬细胞表达炎性介质而发挥其体外抗炎作用,且其下调作用呈剂量依赖性。     

HIF-1α/BNIP3信号通路对BCG诱导巨噬细胞自噬的影响

【背景】缺氧诱导因子1α(hypoxia-inducible factor 1-alpha,HIF-1α)是响应细胞低氧反应的关键因子,在红细胞生成、血管形成、能量代谢及调节宿主免疫代谢中发挥着重要作用。【目的】探讨HIF-1α/Bcl-2-腺病毒E1B相互作用蛋白3(Bcl-2-adenovirus E1B 19-kDa interacting protein 3,BNIP3)信号通路对牛分枝杆菌卡介苗(Bacillus Calmette-Guérin,BCG)诱导小鼠巨噬细胞RAW 264.7自噬的影响。【方法】构建HIF-1α的小干扰RNA (siHIF-1α),转染RAW 264.7细胞后,结合BCG感染,采用流式细胞仪检测细胞自噬率,用Western blotting或免疫荧光技术检测HIF-1α、BNIP3、LC3、Beclin 1、Rheb和mTOR的表达水平。【结果】BCG感染显著上调巨噬细胞中LC3和HIF-1α的表达,用siHIF-1α结合BCG感染后显著下调巨噬细胞中HIF-1α、BNIP3、LC3、Beclin 1和细胞自噬率水平,并促进Rheb和p-mTOR的表达。【结论】在BCG感染RAW 264.7细胞过程中,干扰HIF-1α表达抑制了HIF-1α/BNIP3信号通路,进而激活了mTOR途径,抑制BCG感染诱导的细胞自噬。     

蜂蛹多肽对巨噬细胞RAW264.7免疫活性的影响

蜂蛹多肽因具有丰富的营养价值,以及增强免疫、抗肿瘤及抗氧化等生物学活性,而受到了广泛关注,但目前关于蜂蛹多肽纯化组分的体外免疫调节活性的研究尚未见报道。为了探究蜂蛹多肽对巨噬细胞RAW264.7免疫活性的影响,以蜂蛹多肽纯化组分BPP-21为研究对象,研究其在不同浓度（12.5、25、50、100和200 μg·mL-1）下对RAW264.7巨噬细胞的细胞活力、吞噬能力、细胞因子分泌能力、NO分泌能力和氧化应激指标的影响。结果显示,在浓度12.5~200 μg·mL-1范围内,BPP-21对RAW264.7巨噬细胞无明显的细胞毒性作用,可显著提高干扰素-γ（interferon-gamma,IFN-γ）与NO的分泌水平（P<0.05）;在浓度25~200 μg·mL-1范围内,显著增加细胞吞噬能力以及白细胞介素-2（interleukin-2,IL-2）、肿瘤坏死因子α（tumor necrosis factor-alpha,TNF-α）的分泌量（P<0.05）;在浓度50~200 μg·mL-1范围内,显著提高细胞内超氧化物歧化酶（superoxide dismutase,SOD）活力（P<0.05）。研究表明,蜂蛹多肽纯化组分BPP-21可增强RAW264.7巨噬细胞的免疫活性,为蜂蛹多肽免疫调节剂的研究与开发提供了理论依据。     

酸枣叶醇提物对小鼠巨噬细胞M1和M2型极化的影响

为探究酸枣叶醇提物(jujube leaves alcohol extract,JLE)对RAW264.7细胞M1和M2型极化的影响。本研究分别采用LPS和IL-4将RAW264.7细胞诱导极化为M1和M2型巨噬细胞,CCK8方法检测细胞存活率,Griess法检测药物对M1型细胞释放NO的影响,RT-PCR法检测药物对M1和M2型细胞标志基因表达的影响,划痕实验和RT-PCR法检测经药物处理的M2型巨噬细胞上清对4T1细胞迁移的影响。结果显示,酸枣叶醇提物可抑制NO的释放,下调M1型IL-12p40、IL-1β、COX-2和M2型Arg1、CD206的表达。并通过M2型巨噬细胞降低4T1细胞中MMP-2、MMP-9和NF-κBp65的mRNA水平,抑制4T1细胞的迁移。综上说明酸枣叶醇提物可抑制巨噬细胞M1和M2型极化,并通过NF-κB通路抑制4T1细胞迁移。     

Fucoidan对巨噬细胞RAW264.7的免疫调节作用

目的 观察褐藻多糖硫酸酯（Fucoidan）对巨噬细胞RAW264.7体外吞噬活性、细胞因子TNF-α和IL-6分泌,以及Toll样受体4（TLR4）mRNA表达的影响。方法 实验分对照组,Fucoidan高、中、低剂量组（浓度分别是200、400和800 μg/mL）。药物处理6~48 h后,MTT法检测RAW264.7细胞活力;中性红比色法检测细胞吞噬活性;ELISA法检测培养上清中TNF-α和IL-6的分泌水平;实时定量PCR检测Toll样受体4（TLR4）mRNA表达量。结果 与对照组相比,Fucoidan显著增强RAW264.7细胞代谢活力和吞噬能力（P＜0.01）,增加TNF-α和IL-6的分泌,上调TLR4的表达,呈剂量依赖关系。结论 Fucoidan可上调TLR4表达,增强巨噬细胞代谢和吞噬活性,增加TNF-α和IL-6的分泌,具有潜在的调节免疫作用。     

小鼠肺脏间质树突状细胞形态学分布与观察

目的认识小鼠肺脏间质树突状细胞(dendritic cells,DC)的形态、数量和分布特点。方法运用光镜、电镜观察与免疫组化标记(CD11c、CD205、CD80、CD86及MHC-II类分子I-Ab)方法,观察研究C57BL/6小鼠肺脏间质树突状细胞的形态、数量和分布。结果小鼠肺脏间质树突状细胞具有与免疫器官及外周血中DC相同的超微形态特征;CD11c阳性DC含量约占肺脏总细胞数的8.5‰,广泛分布于肺间质中;表达CD205、CD80、CD86及I-Ab的成熟DC含量稀少,多位于间质血管周围。结论肺脏间质树突状细胞是一种脏器免疫前哨细胞,是肺脏免疫微环境的重要组成部分。     

Nystatin-induced nitric oxide production in mouse macrophage-like cell line RAW264.7

Nystatin is known to deplete lipid rafts from mammalian cell membranes. Lipid rafts have been reported to be necessary for lipopolysaccharide signaling. In this study, it was unexpectedly found that lipopolysaccharide-induced nitric oxide production was not inhibited, but rather increased in the presence of a non-cytotoxic concentration of nystatin. Surprisingly, treatment with nystatin induced only NO production and iNOS expression in RAW264.7 cells. At the concentration used, no changes in the expression of GM1 ganglioside, a lipid raft marker on RAW264.7 cells, was seen. From studies using several kinds of inhibitors for signaling molecules, nystatin-induced NO production seems to occur via the iκB/NF-κB and the PI3 K/Akt pathway. Furthermore, because nystatin is known to activate the Na-K pump, we examined whether the Na-K pump inhibitor amiloride suppresses nystatin-induced NO production. It was found that amiloride significantly inhibited nystatin-induced NO production. The results suggest that a moderate concentration of nystatin induces NO production by Na-pump activation through the PI3 kinase/Akt/NF-κB pathway without affecting the condition of lipid rafts.     

Peroxiredoxin-1, a possible target in modulating inflammatory cytokine production in macrophage like cell line RAW264.7

Peroxiredoxin (PRX), a scavenger of H₂O₂ and alkyl hydroperoxides in living organisms, protects cells from oxidative stress. Contrary to its known anti‐oxidant roles, the involvement of PRX‐1 in the regulation of lipopolysaccharide (LPS) signaling is poorly understood, possible immunological functions of PRX‐1 having been uncovered only recently. In the present study, it was discovered that the PRX‐1 deficient macrophage like cell line (RAW264.7) has anti‐inflammatory activity when stimulated by LPS. Treatment with LPS for 3 hrs resulted in increased gene expression of an anti‐inflammatory cytokine, interleukin‐10 (IL‐10), in PRX‐1 knock down RAW264.7 cells. Gene expression of pro‐inflammatory cytokines IL‐1β and tumor necrosis factor‐ α (TNF‐α) did not show notable changes under the same conditions. However, production of these cytokines significantly decreased in PRX‐1 knock down RAW264.7 cells with 12 hrs of stimulation. Production of IL‐10 was also increased in PRX‐1 knock down RAW264.7 cells with 12 hrs of stimulation. We predicted that higher concentrations of IL‐10 would result in decreased expression of IL‐1β and TNF‐α in PRX‐1 knock‐down cells. This was confirmed by blocking IL‐10, which reestablished IL‐1β and TNF‐α secretion. We also observed that increased concentrations of IL‐10 do not affect the NF‐κB pathway. Interestingly, STAT3 phosphorylation by LPS stimulation was significantly increased in PRX‐1 knockdown RAW264.7 cells. Up‐regulation of IL‐10 in PRX‐1 knockdown cells and the resulting downregulation of proinflammatory cytokine production seem to involve the STAT3 pathway in macrophages. Thus, down‐regulation of PRX‐1 may contribute to the suppression of adverse effects caused by excessive activation of macrophages through affecting the STAT3 signaling pathway.     

Mitochondria are involved in apoptosis induced by ultraviolet radiation in lepidopteran Spodoptera litura cell line

Abstract Mitochondria are involved in apoptosis of mammalian cells and even single‐cell organisms, but mitochondria are not required in apoptosis in cultured Drosophila cells such as S2 and BG2 cell lines. It is not very clear whether mitochondria are involved in apoptosis in other insect cells such as lepidopteran cell lines. Thus, we determined to elucidate the role of mitochondria in apoptosis induced by ultraviolet radiation in Spodoptera litura (Lepidoptera: Noctuidae) cell line (SL‐ZSU‐1). The Western blot results suggested that cytochrome c in the ultraviolet‐treated SL‐1 cells was released from the mitochondria to cytosol as early as 4 h after the induction of ultraviolet radiation and increased in the cytosolic fractions in a time‐dependent manner. Flow cytometric analysis of mitochondrial membrane potential (ΔΨm) of SL‐ZSU‐1 cell treated with ultraviolet‐C (UV‐C) light indicated the decrease in mitochondrial membrane potential was dependent on the times of ultraviolet treatment. Both of them are different from apoptosis in cultured Drosophila melanogaster cell lines (S2 and BG2) and it appears evident mitochondria are involved in apoptosis of the studied lepidopteran cells.     

Transglutaminase overexpression sensitizes neuronal cell lines to apoptosis by increasing mitochondrial membrane potential and cellular oxidative stress

'Tissue' transglutaminase (tTG) selectively accumulates in cells undergoing apoptosis both in vivo and in vitro. Considering the central role played by mitochondria in apoptosis, we investigated the relationships existing amongst tTG expression, apoptosis and mitochondrial function. To this aim we studied the mechanisms of apoptosis in a neuronal cell line (SK-N-BE (2)) in which the tTG-expression was driven by a constitutive promoter. Furthermore, a tet-off inducible promoter was also used in 3T3 fibroblastic cells used as control. Both cell lines, when expressing tTG, appeared 'sensitized' to apoptosis. Strikingly, we found major differences in the morphological features of mitochondria among cell lines in the absence of apoptotic stimuli. In addition, these ultrastructural characteristics were associated with specific functional features: (i) constitutively hyperpolarized mitochondria and (ii) increased reactive oxygen intermediates production. Importantly, after mitochondrial-mediated apoptosis by staurosporine, a rapid loss of mitochondrial membrane potential was found in tTG cells only. Taken together, these results seem to suggest that, via hyperpolarization, tTG might act as a 'sensitizer' towards apoptotic stimuli specifically targeted to mitochondria. These results could also be of pathogenetic relevance for those diseases that are characterized by increased tTG and apoptotic rate together with impaired mitochondrial function, e.g. in some neurodegenerative disease.     

Glucose deprivation induces mitochondrial dysfunction and oxidative stress in PC12 cell line

Glucose metabolism plays a pivotal role in many physiological and pathological conditions. To investigate the effect of hypoglycemia (obtained by glucose deprivation) on PC12 cell line, we analyzed the cell viability, mitochondrial function (assessed by MTT reduction, cellular ATP level, mitochondrial transmembrane potential), and the level of reactive oxygen species (ROS) after glucose deprivation (GD). Upon exposure to GD, ROS level increased and MTT reduction decreased immediately, intracellular ATP level increased in the first 3 hours, followed by progressive decrease till the end of GD treatment, and the mitochondrial transmembrane potential (ΔΨ_m) dropped after 6 hours. Both necrosis and apoptosis occurred apparently after 24 hours which was determined by nuclei staining with propidium iodide(PI) and Hoechst 33342. These data suggested that cytotoxity of GD is mainly due to ROS accumulation and ATP depletion in PC12 cells.     

稳定表达结核分枝杆菌ESAT-6蛋白的RAW264.7细胞系的建立

为研究结核分枝杆菌Mycobacterium tuberculosis分泌蛋白ESAT-6 (Early secreted antigenic target of 6 kDa) 对巨噬细胞相关功能的影响,将正确构建的重组质粒pEGFP-C1-ESAT-6和空载体pEGFP-C1以脂质体介导的方法转染至小鼠巨噬细胞RAW264.7中,经过G418筛选后建立稳定表达EGFP-ESAT6融合蛋白以及EGFP的细胞系,并通过RT-PCR、荧光显微镜及Western blotting方法,在基因和蛋白两个水平对所建立的稳转细胞系进行鉴定。结果证实EGFP-ESAT6融合基因成功整合入RAW264.7细胞基因组并能够稳定表达,为后续的ESAT-6调控巨噬细胞机理研究提供了平台。     

Mechanisms of B cell receptor induced apoptosis

B cell receptor (BCR)-mediated apoptosis plays a key role in the negative selection (deletion) of autoreactive B cells. Mechanisms of BCR-mediated apoptosis have been widely studied in cell lines representing both immature (bone marrow) and mature (germinal center) B cells. However, there is much inconsistency and controversy concerning the possible mechanisms of BCR-mediated apoptosis, which may reflect differences in the origin or the maturational stage of the cell line used. Based on recent studies, collapse of mitochondrial membrane potential (Delta Psi m) seems to be an essential event for BCR-mediated apoptosis in both mature and immature cells. The collapse of Delta Psi m is dependent on the synthesis of new proteins, which are involved in the permeability change of mitochondrial membranes. Mitochondrial dysfunction induces activation of caspases, cysteine proteases, which play a central role in apoptosis. However, instead of caspases, other effector proteases, such as cathepsins or calpains, may also be responsible for the organized destruction of cell components seen during BCR-mediated apoptosis.